Cell

ABSTRACT

The present invention provides a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising an antigen-binding domain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22.

FIELD OF THE INVENTION

The present invention relates to a cell which comprises more than onechimeric antigen receptor (CAR).

BACKGROUND TO THE INVENTION

A number of immunotherapeutic agents have been described for use incancer treatment, including therapeutic monoclonal antibodies (mAbs),immunoconjugated mAbs, radioconjugated mAbs and bi-specific T-cellengagers.

Typically these immunotherapeutic agents target a single antigen: forinstance, Rituximab targets CD20; Myelotarg targets CD33; andAlemtuzumab targets CD52.

The human CD19 antigen is a 95 kd transmembrane glycoprotein belongingto the immunoglobulin superfamily. CD19 is expressed very early inB-cell differentiation and is only lost at terminal B-celldifferentiation into plasma cells. Consequently, CD19 is expressed onall B-cell malignancies apart from multiple myeloma. Since loss of thenormal B-cell compartment is an acceptable toxicity, CD19 is anattractive CAR target and clinical studies targeting CD19 with CARs haveseen promising results.

A particular problem in the field of oncology is provided by theGoldie-Coldman hypothesis: which describes that the sole targeting of asingle antigen may result in tumour escape by modulation of said antigendue to the high mutation rate inherent in most cancers. This modulationof antigen expression may reduce the efficacy of knownimmunotherapeutics, including those which target CD19.

Thus a problem with immunotherapeutics targeted against CD19 is that aB-cell malignancy may mutate and become CD19-negative. This may resultin relapse with CD19-negative cancers which are not responsive to CD19targeted therapeutics. For example, in one paediatric study, Grupp etal. reported that half of all relapses following CD19-targeted chimericantigen receptor therapy for B-acute Lymphoblastic leukaemia (B-ALL)were due to CD19-negative disease (56^(th) American Society ofHematology Annual Meeting and Exposition).

There is thus a need for immunotherapeutic agents which are capable oftargeting more than one cell surface structure to reflect the complexpattern of marker expression that is associated with many cancers,including CD19-positive cancers.

Chimeric Antigen Receptors (CARs)

Chimeric antigen receptors are proteins which graft the specificity of,for example, a monoclonal antibody (mAb) to the effector function of aT-cell. Their usual form is that of a type I transmembrane domainprotein with an antigen recognizing amino terminus, a spacer, atransmembrane domain all connected to a compound endodomain whichtransmits T-cell survival and activation signals (see FIG. 1A).

The most common form of these molecules are fusions of single-chainvariable fragments (scFv) derived from monoclonal antibodies whichrecognize a target antigen, fused via a spacer and a trans-membranedomain to a signaling endodomain. Such molecules result in activation ofthe T-cell in response to recognition by the scFv of its target. When Tcells express such a CAR, they recognize and kill target cells thatexpress the target antigen. Several CARs have been developed againsttumour associated antigens, and adoptive transfer approaches using suchCAR-expressing T cells are currently in clinical trial for the treatmentof various cancers.

It has been observed that using a CAR approach for cancer treatment,tumour heterogeneity and immunoediting can cause escape from CARtreatment. For example, in the study described by Grupp et al (2013; NewEng. J. Med 368:1509-1518, paper No 380, ASH 2014) CAR-modified T cellapproach was used for the treatment of acute B-lymphocytic leukemia. Inthat clinical trial it was found that 10 patients with a completeremission after one month did relapse and 5 of them relapsed withCD19-negative disease.

There is thus a need for alternative CAR treatment approaches whichaddress the problems of cancer escape and tumour heterogeneity.

Expression of Two CAR Binding Specificities

Bispecific CARs known as tandem CARs or TanCARs have been developed inan attempt to target multiple cancer specific markers simultaneously. Ina TanCAR, the extracellular domain comprises two antigen bindingspecificities in tandem, joined by a linker. The two bindingspecificities (scFvs) are thus both linked to a single transmembraneportion: one scFv being juxtaposed to the membrane and the other beingin a distal position.

Grada et al (2013, Mol Ther Nucleic Acids 2:e105) describes a TanCARwhich includes a CD19-specific scFv, followed by a Gly-Ser linker andthen a HER2-specific scFv. The HER2-scFv was in the juxta-membraneposition, and the CD19-scFv in the distal position. The Tan CAR wasshown to induce distinct T cell reactivity against each of the twotumour restricted antigens. This arrangement was chosen because therespective lengths of HER2 (632 aa/125A) and CD19 (280aa, 65A) lendsitself to that particular spatial arrangement. It was also known thatthe HER2 scFv bound the distal-most 4 loops of HER2.

The problem with this approach is that the juxta-membrane scFv may beinaccessible due to the presence of the distal scFv, especially which itis bound to the antigen. In view of the need to choose the relativepositions of the two scFvs in view of the spatial arrangement of theantigen on the target cell, it may not be possible to use this approachfor all scFv binding pairs. Moreover, it is unlikely that the TanCarapproach could be used for more than two scFvs, a TanCAR with three ormore scFvs would be a very large molecule and the scFvs may well foldback on each other, obscuring the antigen-binding sites. It is alsodoubtful that antigen-binding by the most distal scFv, which isseparated from the transmembrane domain by two or more further scFvs,would be capable of triggering T cell activation.

There is thus a need for an alternative approach to express two CARbinding specificities on the surface of a cell such as a T cell.

SUMMARY OF THE INVENTION

The present inventors have developed a CAR T cell which expresses twoCARs at the cell surface, one specific for CD19 and one specific forCD22.

Thus in a first aspect the present invention provides a cell whichco-expresses a first chimeric antigen receptor (CAR) and second CAR atthe cell surface, each CAR comprising an antigen-binding domain, whereinthe antigen-binding domain of the first CAR binds to CD19 and theantigen-binding domain of the second CAR binds to CD22.

The fact the one CAR binds CD19 and the other CAR binds CD22 isadvantageous because some lymphomas and leukaemias become CD19 negativeafter CD19 targeting, (or possibly CD22 negative after CD22 targeting),so it gives a “back-up” antigen, should this occur.

The cell may be an immune effector cell, such as a T-cell or naturalkiller (NK) cell. Features mentioned herein in connection with a T cellapply equally to other immune effector cells, such as NK cells.

Each CAR may comprise:

-   -   (i) an antigen-binding domain;    -   (ii) a spacer; and    -   (iii) a trans-membrane domain.

Each CAR may comprise:

-   -   (i) an antigen-binding domain;    -   (ii) a spacer;    -   (iii) a trans-membrane domain;    -   (iv) an endodomain.

The spacer of the first CAR may be different to the spacer of the secondCAR, such the first and second CAR do not form heterodimers.

The spacer of the first CAR may have a different length and/orconfiguration from the spacer of the second CAR, such that each CAR istailored for recognition of its respective target antigen.

The antigen-binding domain of the second CAR may bind to amembrane-distal epitope on CD22. The antigen-binding domain of thesecond CAR may bind to an epitope on Ig domain 1, 2, 3 or 4 of CD22, forexample on Ig domain 3 of CD22.

The antigen-binding domain of the first CAR may bind to an epitope onCD19 which is encoded by exon 1, 3 or 4.

The endodomain of one CAR may comprise a co-stimulatory domain and anITAM-containing domain; and the endodomain of the other CAR may comprisea TNF receptor family domain and an ITAM-containing domain.

For example, one CAR (which may be CD19 or CD22-specific) may have thestructure:

AgB1-spacer1-TM1-costim-ITAM

in which:

AgB1 is the antigen-binding domain;

spacer 1 is the spacer;

TM1 is the transmembrane domain;

costim is a co-stimulatory domain; and

ITAM is an ITAM-containing endodomain;

and the other CAR (which may be CD22 or CD19-specific) may have thestructure:

-   -   AgB2-spacer2-TM2- TNF-ITAM

in which:

AgB2 is the antigen-binding domain;

spacer 2 is the spacer;

TM2 is the transmembrane domain;

TNF is a TNF receptor endodomain; and

ITAM is an ITAM-containing endodomain.

In a second aspect, the present invention provides, a nucleic acidsequence encoding both the first and second chimeric antigen receptors(CARs) as defined in the first aspect of the invention.

The nucleic acid sequence may have the following structure:

-   -   AgB1-spacer1-TM1 -coexpr-AbB2-spacer2-TM2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a a nucleic acid sequence encoding the transmembrane domain ofthe first CAR;

coexpr is a nucleic acid sequence enabling co-expression of both CARs

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;

TM2 is a a nucleic acid sequence encoding the transmembrane domain ofthe second CAR;

which nucleic acid sequence, when expressed in a T cell, encodes apolypeptide which is cleaved at the cleavage site such that the firstand second CARs are co-expressed at the T cell surface.

The nucleic acid sequence may have the following structure:

-   -   AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a a nucleic acid sequence encoding the transmembrane domain ofthe first CAR;

endo 1 is a nucleic acid sequence encoding the endodomain of the firstCAR;

coexpr is a nucleic acid sequence enabling co-expression of both CARs

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;

TM2 is a a nucleic acid sequence encoding the transmembrane domain ofthe second CAR;

endo 2 is a nucleic acid sequence encoding the endodomain of the secondCAR;

which nucleic acid sequence, when expressed in a T cell, encodes apolypeptide which is cleaved at the cleavage site such that the firstand second CARs are co-expressed at the T cell surface.

The nucleic acid sequence allowing co-expression of two CARs may encodea self -cleaving peptide or a sequence which allows alternative means ofco-expressing two CARs such as an internal ribosome entry sequence or a2^(nd) promoter or other such means whereby one skilled in the art canexpress two proteins from the same vector.

Alternative codons may be used in regions of sequence encoding the sameor similar amino acid sequences, such as the transmembrane and/orintracellular T cell signalling domain (endodomain) in order to avoidhomologous recombination. For example, alternative codons may be used inthe portions of sequence encoding the spacer, the transmembrane domainand/or all or part of the endodomain, such that the two CARs have thesame or similar amino acid sequences for this or these part(s) but areencoded by different nucleic acid sequences.

In a third aspect, the present invention provides kit which comprises

-   -   (i) a first nucleic acid sequence encoding the first chimeric        antigen receptor (CAR), which nucleic acid sequence has the        following structure:    -   AgB1-spacer1-TM1

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR; and

-   -   (ii) a second nucleic acid sequence encoding the second chimeric        antigen receptor, which nucleic acid sequence has the following        structure:    -   AgB2-spacer2-TM2

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR; and

TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR.

The kit may comprise

-   -   (i) a first nucleic acid sequence encoding the first chimeric        antigen receptor (CAR), which nucleic acid sequence has the        following structure:    -   AgB1-spacer1-TM1-endo1

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR;

endo 1 is a nucleic acid sequence encoding the endodomain of the firstCAR; and

-   -   (ii) a second nucleic acid sequence encoding the second chimeric        antigen receptor (CAR), which nucleic acid sequence has the        following structure:    -   AgB2-spacer2-TM2-endo2

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR;

endo 2 is a nucleic acid sequence encoding the endodomain of the secondCAR.

In a fourth aspect, the present invention provides a kit comprising: afirst vector which comprises the first nucleic acid sequence; and asecond vector which comprises the second nucleic acid sequence.

The vectors may be plasmid vectors, retroviral vectors or transposonvectors. The vectors may be lentiviral vectors.

In a fifth aspect, the present invention provides a vector comprising anucleic acid sequence according to the second aspect of the invention.The vector may be a lentiviral vector.

The vector may be a plasmid vector, a retroviral vector or a transposonvector.

In a sixth aspect the present invention provides a method for making acell according to the first aspect of the invention, which comprises thestep of introducing one or more nucleic acid sequence(s) encoding thefirst and second CARs; or one or more vector(s), as defined above, intoa T cell.

The cell may be from a sample isolated from a patient, a related orunrelated haematopoietic transplant donor, a completely unconnecteddonor, from cord blood, differentiated from an embryonic cell line,differentiated from an inducible progenitor cell line, or derived from atransformed cell line.

In a seventh aspect, the present invention provides a pharmaceuticalcomposition comprising a plurality of cells according to the firstaspect of the invention.

In an eighth aspect the present invention provides a method for treatingand/or preventing a disease, which comprises the step of administering apharmaceutical composition according to the seventh aspect of theinvention to a subject.

The method may comprise the following steps:

-   -   (i) isolation of a cell-containing sample from a subject;    -   (ii) transduction or transfection of the cells with one or more        nucleic acid sequence(s) encoding the first and second CAR or        one or more vector(s) comprising such nucleic acid sequence(s);        and    -   (iii) administering the cells from (ii) to a the subject.

The disease may be cancer. The cancer may be a B cell malignancy.

In a ninth aspect the present invention provides a pharmaceuticalcomposition according to the seventh aspect of the invention for use intreating and/or preventing a disease.

In a tenth aspect the present invention provides the use of a cellaccording to the first aspect of the invention in the manufacture of amedicament for treating and/or preventing a disease.

The present invention also provides a nucleic acid sequence whichcomprises:

a) a first nucleotide sequence encoding a first chimeric antigenreceptor (CAR);

b) a second nucleotide sequence encoding a second CAR; wherein one CARbinds CD19 and the other CAR binds CD22; and

c) a sequence encoding a self-cleaving peptide positioned between thefirst and second nucleotide sequences, such that the two CARs areexpressed as separate entities.

Alternative codons may be used in one or more portion(s) of the firstand second nucleotide sequences in regions which encode the same orsimilar amino acid sequence(s).

The present invention also provides a vector and a cell comprising sucha nucleic acid.

The present inventors have also developed new anti-CD19 and anti-CD22CARs with improved properties.

Thus in an eleventh aspect, the present invention provides a chimericantigen receptor (CAR) comprising a CD19-binding domain which comprises

a) a heavy chain variable region (VH) having complementarity determiningregions (CDRs) with the following sequences:

(SEQ ID No. 15) CDR1-SYWMN; (SEQ ID No. 16) CDR2-QIWPGDGDTNYNGKFK(SEQ ID No. 17) CDR3-RETTTVGRYYYAMDY;and

b) a light chain variable region (VL) having CDRs with the followingsequences:

(SEQ ID No. 18) CDR1-KASQSVDYDGDSYLN; (SEQ ID No. 19) CDR2-DASNLVS(SEQ ID No. 20) CDR3-QQSTEDPWT.

The CD19 binding domain may comprise a VH domain having the sequenceshown as SEQ ID No. 23, or SEQ ID NO 24; or a VL domain having thesequence shown as SEQ ID No 25, SEQ ID No. 26 or SEQ ID No. 40 or avariant thereof having at least 90% sequence identity which retains thecapacity to bind CD19.

The CD19 binding domain may comprise the sequence shown as SEQ ID No 21,SEQ ID No. 22 or SEQ ID No. 39 or a variant thereof having at least 90%sequence identity which retains the capacity to bind CD19.

In a twelfth aspect the present invention provides a chimeric antigenreceptor (CAR) comprising a CD22-binding domain which comprises

a) a heavy chain variable region (VH) having complementarity determiningregions (CDRs) with the followina seauences:

(SEQ ID No. 27) CDR1-NYWIN; (SEQ ID No. 28) CDR2-NIYPSDSFTNYNQKFKD(SEQ ID No. 29) CDR3-DTQERSWYFDV;and

b) a light chain variable region (VL) having CDRs with the followingsequences:

(SEQ ID No. 30) CDR1-RSSQSLVHSNGNTYLH; (SEQ ID No. 31) CDR2-KVSNRFS(SEQ ID No. 32) CDR3-SQSTHVPWT.

The CD22 binding domain may comprise a VH domain having the sequenceshown as SEQ ID No. 35, or SEQ ID NO 36; or a VL domain having thesequence shown as SEQ ID No 37, or SEQ ID No. 38 or a variant thereofhaving at least 90% sequence identity which retains the capacity to bindCD22.

The CD22 binding domain may comprise the sequence shown as SEQ ID No 33or SEQ ID No. 34 or a variant thereof having at least 90% sequenceidentity which retains the capacity to bind CD22.

In a thirteenth aspect there is provided a cell which expresses achimeric antigen receptor according to the eleventh aspect of theinvention or a chimeric antigen receptor according to the twelfth aspectof the invention at the cell surface.

In a fourteenth aspect, there is provided a nucleic acid sequenceencoding a chimeric antigen receptor according to the eleventh aspect ofthe invention or a chimeric antigen receptor according to the twelfthaspect of the invention.

In a fifteenth aspect, the present invention provides a vectorcomprising a nucleic acid sequence according to the fourteenth aspect ofthe invention. The vector may be a lentiviral vector.

The vector may be a plasmid vector, a retroviral vector or a transposonvector.

In a sixteenth aspect, the present invention provides a method formaking a cell according to the thirteenth aspect of the invention, whichcomprises the step of introducing one or more nucleic acid sequence(s);or one or more vector(s), as defined above, into a cell.

The cell may be a T-cell or a natural killer (NK) cell. The cell may befrom a sample isolated from a patient, a related or unrelatedhaematopoietic transplant donor, a completely unconnected donor, fromcord blood, differentiated from an embryonic cell line, differentiatedfrom an inducible progenitor cell line, or derived from a transformedcell line.

In a seventeenth aspect, the present invention provides a pharmaceuticalcomposition comprising a plurality of cells according to the thirteenthaspect of the invention.

In an eighteenth aspect the present invention provides a method fortreating and/or preventing a disease, which comprises the step ofadministering a pharmaceutical composition according to the seventeenthaspect of the invention to a subject.

The method may comprise the following steps:

-   -   (i) isolation of a cell-containing sample from a subject;    -   (ii) transduction or transfection of the cells with a nucleic        acid sequence encoding the CAR or a vector comprising such a        nucleic acid sequence; and    -   (iii) administering the cells from (ii) to a the subject.

The disease may be cancer. The cancer may be a B cell malignancy.

In a ninteenth aspect the present invention provides a pharmaceuticalcomposition according to the seventeenth aspect of the invention for usein treating and/or preventing a disease.

In a twentieth aspect the present invention provides the use of a cellaccording to the thirteenth aspect of the invention in the manufactureof a medicament for treating and/or preventing a disease.

There is also provided a cell according to the first aspect of theinvention, which comprises a first CAR as defined in the eleventh aspectof the invention and a second CAR as defined in the twelfth aspect ofthe invention.

There is also provided a nucleic acid sequence according to the secondaspect of the invention, encoding a first CAR as defined in the eleventhaspect of the invention and a second CAR as defined in the twelfthaspect of the invention.

There is also provided a kit according to the third aspect of theinvention, wherein the first nucleic acid sequence encodes a first CARas defined in the eleventh aspect of the invention and the secondnucleic acid sequence encodes a second CAR as defined in the twelfthaspect of the invention.

There is also provided a vector according to the fifth aspect of theinvention, which comprises a nucleic acid sequence encoding a first CARas defined in the eleventh aspect of the invention and a second CAR asdefined in the twelfth aspect of the invention.

The present inventors have also found that, in an OR gate system,performance is improved if the co-stimulatory domain and domainproducing survival signals are “split” between the two (or more) CARs.

Thus, in a twenty-first aspect there is provided a cell whichco-expresses a first chimeric antigen receptor (CAR) and second CAR atthe cell surface, each CAR comprising an intracellular signallingdomain, wherein the intracellular signalling domain of the first CARcomprises a co-stimulatory domain; and the intracellular signallingdomain of the second CAR comprises a TNF receptor family endodomain.

The co-stimulatory domain may be a CD28 co-stimulatory domain.

The TNF receptor family endodomain may be, for example OX-40 or 4-1BBendodomain.

The intracellular signalling domain of the first and the second CAR mayalso comprise an ITAM-containing domain, such as a CD3 zeta endodomain.

The first CAR may have the structure:

-   -   AgB1-spacer1-TM1-costim-ITAM

in which:

AgB1 is the antigen-binding domain of the first CAR;

spacer 1 is the spacer of the first CAR;

TM1 is the transmembrane domain of the first CAR;

costim is a co-stimulatory domain; and

ITAM is an ITAM-containing endodomain.

The second CAR may have the structure:

-   -   AgB2-spacer2-TM2-TNF-ITAM

in which:

AgB2 is the antigen-binding domain of the second CAR;

spacer 2 is the spacer of the second CAR;

TM2 is the transmembrane domain of the second CAR;

TNF is a TNF receptor endodomain; and

ITAM is an ITAM-containing endodomain.

One CAR out of the first and second CAR may target CD19 and the otherCAR may target CD22.

In a twenty-second aspect there is provided a nucleic acid sequenceencoding both the first and second chimeric antigen receptors (CARs) asdefined in the twenty-first aspect of the invention.

The nucleic acid sequence may have the following structure:

-   -   AgB1-spacer1-TM1-costim-ITAM1-coexpr-AbB2-spacer2-TM2-TNF-ITAM2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a a nucleic acid sequence encoding the transmembrane domain ofthe first CAR;

costim is a nucleic acid sequence encoding a co-stimulatory domain;

ITAM1 is a nucleic acid sequence encoding the ITAM-containing endodomainof the first CAR;

coexpr is a nucleic acid sequence enabling co-expression of both CARs

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR;

TNF is a nucleic acid sequence encoding a TNF receptor endodomain;

ITAM2 is a nucleic acid sequence encoding the ITAM-containing endodomainof the second CAR.

When the nucleic acid sequence is expressed in a cell it may encode apolypeptide which is cleaved at the cleavage site such that the firstand second CARs are co-expressed at the cell surface.

In a twenty-third aspect, there is provided a kit which comprises

-   -   (i) a first nucleic acid sequence encoding the first chimeric        antigen receptor (CAR) as defined in the twenty-first aspect of        the invention, which nucleic acid sequence has the following        structure:    -   AgB1-spacer1-TM1-costim-ITAM1

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR;

costim is a nucleic acid sequence encoding a co-stimulatory domain;

ITAM1 is a nucleic acid sequence encoding the ITAM-containing endodomainof the first CAR;

and

-   -   (ii) a second nucleic acid sequence encoding the second chimeric        antigen receptor (CAR) as defined in the twenty-first aspect of        the invention, which nucleic acid sequence has the following        structure:    -   AbB2-spacer2-TM2-TNF-ITAM2

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR;

TNF is a nucleic acid sequence encoding a TNF receptor endodomain; and

ITAM2 is a nucleic acid sequence encoding the ITAM-containing endodomainof the second CAR.

In a twenty-fourth aspect there is provided a vector comprising anucleic acid sequence according to the twenty-second aspect of theinvention or as defined in the twenty-third aspect of the invention.

In a twenty-fifth aspect, there is provided a method for making a cellaccording to the twenty-first aspect of the invention, which comprisesthe step of introducing: a nucleic acid sequence according totwenty-second aspect of the invention; a first nucleic acid sequence anda second nucleic acid sequence as defined in the twenty-third aspect ofthe invention; or a vector according to the twenty-fourth aspect of theinvention, into a cell.

In a twenty-sixth aspect, the present invention provides apharmaceutical composition comprising a plurality of cells according tothe twenty-first aspect of the invention.

There is also provided a method for treating and/or preventing adisease, which comprises the step of administering a pharmaceuticalcomposition according to the twenty-sixth aspect of the invention to asubject.

There is also provided a pharmaceutical composition according to thetwenty-sixth aspect of the invention for use in treating and/orpreventing a disease.

There is also provided the use of a cell according to the twenty-firstaspect of the invention in the manufacture of a medicament for treatingand/or preventing a disease.

By providing one CAR which targets CD19 and one CAR which targets CD22,it is possible to target each of these markers, thereby reducing theproblem of cancer escape.

Because the CARs are expressed on the surface of the cell as separatemolecules, this approach overcomes the spatial and accessibility issuesassociated with TanCARs. Cell activation efficiency is also improved. Ifeach CAR has its own spacer, it is possible to tailor the spacer andtherefore the distance that the binding domain projects from the cellsurface and its flexibility etc. to the particular target antigen. Thischoice is unfettered by the design considerations associated withTanCARs, i.e. that one CAR needs to be juxtaposed to the T cell membraneand one CAR needs to be distal, positioned in tandem to the first CAR.

By providing a single nucleic acid which encodes the two CARs separatedby a cleavage site, it is possible to engineer cells to co-express thetwo CARs using a simple single transduction procedure. A doubletransfection procedure could be used with CAR-encoding sequences inseparate constructs, but this would be more complex and expensive andrequires more integration sites for the nucleic acids. A doubletransfection procedure would also be associated with uncertainty as towhether both CAR-encoding nucleic acids had been transduced andexpressed effectively.

The CARs will have portions of high homology, for example thetransmembrane and/or intracellular signalling domains are likely to behighly homologous. If the same or similar linkers are used for the twoCARs, then they will also be highly homologous. This would suggest thatan approach where both CARs are provided on a single nucleic acidsequence would be inappropriate, because of the likelihood of homologousrecombination between the sequences. However, the present inventors havefound that by “codon wobbling” the portions of sequence encoding areasof high homology, it is possible to express two CARs from a singleconstruct with high efficiency. Codon wobbling involves usingalternative codons in regions of sequence encoding the same or similaramino acid sequences.

DESCRIPTION OF THE FIGURES

FIG. 1: a) Schematic diagram illustrating a classical CAR. (b) to (d):Different generations and permutations of CAR endodomains: (b) initialdesigns transmitted ITAM signals alone through FceR1-γ or CD3endodomain, while later designs transmitted additional (c) one or (d)two co-stimulatory signals in the same compound endodomain.

FIG. 2: B-cell maturation pathway/B-cell ontogeny. DR=HLA-DR;cCD79=cytoplasmic CD79; cCD22 =cytoplasmic CD22. Both CD19 and CD22antigens are expressed during early stages in B-cell maturation. It isthese cells that develop into B-cell acute leukaemias. Targeting bothCD19 as well as CD22 simultaneously is most suited for targeting B-cellacute leukaemias.

FIG. 3: Strategies for design of an anti-CD19 OR CD22 CAR cassette.Binders which recognize CD19 and binders which recognize CD22 areselected. An optimal spacer domain and signalling domain is selected foreach CAR. (a) an OR gate cassette is constructed so that both CARs areco-expressed using a FMD-2A peptide. Any homologous sequences arecodon-wobbled to avoid recombination. (c) The two CARs are co-expressedas separate proteins on the T-cell surface.

FIG. 4: Example of codon-wobbling to allow co-expression in a retroviralvector of identical peptide sequences but avoiding homologousrecombination. Here, wild-type HCH2CH3-CD28tmZeta is aligned withcodon-wobbled HCH2CH3-CD28tmZeta.

FIG. 5: Demonstrating functionality of anti-CD19 OR CD22 CAR gate. (a)Cartoon of construct: S1—signal peptide 1; HA—haemagglutin tag;HCH2CH3—hinge, CH2CH3 of IgG1 wild-type sequence; CD28tmZ—CD28transmembrane domain and CD3 Zeta wobbled sequence; 2A—Foot and mouthdisease 2A peptide; S2—signal peptide 2; V5—v5 epitope tag;aCD22—anti-CD22 scFv; HCH2CH3′—hinge, CH2CH3 of IgG1 wobbled sequence;CD28tmZ—CD28 transmembrane domain and CD3 Zeta wobbled sequence.; (b)Co-expression of two receptors from a single vector. Peripheral bloodT-cells were transduced with bicistronic vector after stimulation withOKT3 and anti-CD28. Cells were analysed five days after transduction bystaining with anti-V5-FITC (invitrogen) and anti-HA-PE (abCam). The twoCARs can be detected simultaneously on the T-cell surface. (c)Non-transduced T-cells, T-cells expressing just anti-CD19 CAR, T-cellsexpressing just anti-CD22 CAR and T-cells expressing the anti-CD19 ORCD22 CAR gate were challenged with target cells expressing neither CD19or CD22, either CD19 or CD22 singly, or both antigen. T-cells expressingthe anti-CD19 OR CD22 CAR gate could kill target cells even if oneantigen was absent.

FIG. 6: Biacore affinity determination for murine CD22ALAb scFv,humanised CD22ALAb scFv and M971 scFv

FIG. 7: Biacore affinity determination for murine CD19ALAb scFv andhumanised CD19ALAb

FIG. 8: Comparison of the binding kinetics between soluble scFv-CD19binding for CD19ALAb scFv and fmc63 scFv

FIG. 9: Schematic diagram illustrating CD19ALAb CAR, fmc63 CAR, CD22ALAbCAR and M971 CAR used in the comparative studies

FIG. 10: Killing assay of CD19 positive target cells comparing a CARwith a CD19ALAb antigen binding domain and an equivalent CAR with anfmc63 binding domain.

FIG. 11: A) Killing assay of CD22 positive target cells comparing a CARwith a CD22ALAb antigen binding domain and an equivalent CAR with anM971 binding domain. B) Assay comparing IFNγ release followingco-culture 1:1 with CD22 positive SupT1 cells

FIG. 12: CD19 structure and exons

FIG. 13: Schematic diagrams and construct maps illustrating the fourconstructs tested in Example 5. In the construct map, portions markedwith are codon-wobbled. A: CD19 and CD22 CAR both have 41BB-CD3zetacompound endodomains; B: CD19 and CD22 CAR both have OX40-CD3zetacompound endodomains; C: CD19 CAR has 41BB-CD3zeta compound endodomainand CD22 CAR has CD28-CD3zeta compound endodomain; and D: CD19 CAR hasOX40-CD3zeta compound endodomain and CD22 CAR has CD28-CD3zeta compoundendodomain

FIG. 14: Target cell killing by cells expressing the constructs shown inFIG. 13.

DETAILED DESCRIPTION Chimeric Antigen Receptors (CARs)

CARs, which are shown schematically in FIG. 1, are chimeric type Itrans-membrane proteins which connect an extracellularantigen-recognizing domain (binder) to an intracellular signallingdomain (endodomain). The binder is typically a single-chain variablefragment (scFv) derived from a monoclonal antibody (mAb), but it can bebased on other formats which comprise an antibody-like antigen bindingsite. A spacer domain is usually necessary to isolate the binder fromthe membrane and to allow it a suitable orientation. A common spacerdomain used is the Fc of IgG1. More compact spacers can suffice e.g. thestalk from CD8a and even just the IgG1 hinge alone, depending on theantigen. A trans-membrane domain anchors the protein in the cellmembrane and connects the spacer to the endodomain.

Early CAR designs had endodomains derived from the intracellular partsof either the y chain of the FcER1 or CD3. Consequently, these firstgeneration receptors transmitted immunological signal 1, which wassufficient to trigger T-cell killing of cognate target cells but failedto fully activate the T-cell to proliferate and survive. To overcomethis limitation, compound endodomains have been constructed: fusion ofthe intracellular part of a T-cell co-stimulatory molecule to that ofCD3 results in second generation receptors which can transmit anactivating and co-stimulatory signal simultaneously after antigenrecognition. The co-stimulatory domain most commonly used is that ofCD28. This supplies the most potent co-stimulatory signal—namelyimmunological signal 2, which triggers T-cell proliferation. Somereceptors have also been described which include TNF receptor familyendodomains, such as the closely related OX40 and 41BB which transmitsurvival signals. Even more potent third generation CARs have now beendescribed which have endodomains capable of transmitting activation,proliferation and survival signals.

CAR-encoding nucleic acids may be transferred to T cells using, forexample, retroviral vectors. Lentiviral vectors may be employed. In thisway, a large number of cancer-specific T cells can be generated foradoptive cell transfer. When the CAR binds the target-antigen, thisresults in the transmission of an activating signal to the T-cell it isexpressed on. Thus the CAR directs the specificity and cytotoxicity ofthe T cell towards tumour cells expressing the targeted antigen.

The first aspect of the invention relates to a cell which co-expresses afirst CAR and a second CAR, wherein one CAR binds CD19 and the other CARbinds CD22, such that a T-cell can recognize a target cells expressingeither of these markers.

Thus, the antigen binding domains of the first and second CARs of thepresent invention bind to different antigens and both CARs may comprisean activating endodomain. The two CARs may comprise spacer domains whichmay be the same, or sufficiently different to prevent cross-pairing ofthe two different receptors. A cell can hence be engineered to activateupon recognition of either or both CD19 and CD22. This is useful in thefield of oncology as indicated by the Goldie-Coldman hypothesis: soletargeting of a single antigen may result in tumour escape by modulationof said antigen due to the high mutation rate inherent in most cancers.By simultaneously targeting two antigens, the probably of such escape isexponentially reduced.

It is important that the two CARs do not heterodimerize.

The first and second CAR of the T cell of the present invention may beproduced as a polypeptide comprising both CARs, together with a cleavagesite.

Signal Peptide

The CARs of the cell of the present invention may comprise a signalpeptide so that when the CAR is expressed inside a cell, such as aT-cell, the nascent protein is directed to the endoplasmic reticulum andsubsequently to the cell surface, where it is expressed.

The core of the signal peptide may contain a long stretch of hydrophobicamino acids that has a tendency to form a single alpha-helix. The signalpeptide may begin with a short positively charged stretch of aminoacids, which helps to enforce proper topology of the polypeptide duringtranslocation. At the end of the signal peptide there is typically astretch of amino acids that is recognized and cleaved by signalpeptidase. Signal peptidase may cleave either during or after completionof translocation to generate a free signal peptide and a mature protein.The free signal peptides are then digested by specific proteases.

The signal peptide may be at the amino terminus of the molecule.

The signal peptide may comprise the SEQ ID No. 1, 2 or 3 or a variantthereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions,substitutions or additions) provided that the signal peptide stillfunctions to cause cell surface expression of the CAR.

SEQ ID No. 1: MGTSLLCWMALCLLGADHADG

The signal peptide of SEQ ID No. 1 is compact and highly efficient. Itis predicted to give about 95% cleavage after the terminal glycine,giving efficient removal by signal peptidase.

SEQ ID No. 2:  MSLPVTALLLPLALLLHAARP

The signal peptide of SEQ ID No. 2 is derived from IgG1.

SEQ ID No. 3:  MAVPTQVLGLLLLWLTDARC

The signal peptide of SEQ ID No. 3 is derived from CD8.

The signal peptide for the first CAR may have a different sequence fromthe signal peptide of the second CAR.

CD19

The human CD19 antigen is a 95 kd transmembrane glycoprotein belongingto the immunoglobulin superfamily. CD19 is classified as a type Itransmembrane protein, with a single transmembrane domain, a cytoplasmicC-terminus, and extracellular N-terminus. The general structure for CD19is illustrated in FIG. 12.

CD19 is a biomarker for normal and neoplastic B cells, as well asfollicular dendritic cells. In fact, it is present on B cells fromearliest recognizable B-lineage cells during development to B-cellblasts but is lost on maturation to plasma cells. It primarily acts as aB cell co-receptor in conjunction with CD21 and CD81. Upon activation,the cytoplasmic tail of CD19 becomes phosphorylated, which leads tobinding by Src-family kinases and recruitment of PI-3 kinase. CD19 isexpressed very early in B-cell differentiation and is only lost atterminal B-cell differentiation into plasma cells. Consequently, CD19 isexpressed on all B-cell malignancies apart from multiple myeloma.

Different designs of CARs have been tested against CD19 in differentcentres, as outlined in the following Table:

TABLE 1 Centre Binder Endodomain Comment University College Fmc63CD3-Zeta Low-level brief persistence London Memorial Sloane SJ25C1CD28-Zeta Short-term persistence Kettering NCI/KITE Fmc63 CD28-ZetaLong-term low-level persistence Baylor, Centre for Fmc63 CD3-Zeta/Short-term low-level Cell and Gene CD28-Zeta persistence TherapyUPENN/Novartis Fmc63 41BB-Zeta Long-term high-level persistence

As shown above, most of the studies conducted to date have used an scFvderived from the hybridoma fmc63 as part of the binding domain torecognize CD19.

As shown in FIG. 12, the gene encoding CD19 comprises ten exons: exons 1to 4 encode the extracellular domain; exon 5 encodes the transmembranedomain; and exons 6 to 10 encode the cytoplasmic domain.

In the CD19/CD22 OR gate of the present invention, the antigen-bindingdomain of the anti-CD19 CAR may bind an epitope of CD19 encoded by exon1 of the CD19 gene.

In the CD19/CD22 OR gate of the present invention, the antigen-bindingdomain of the anti-CD19 CAR may bind an epitope of CD19 encoded by exon3 of the CD19 gene.

In the CD19/CD22 OR gate of the present invention, the antigen-bindingdomain of the anti-CD19 CAR may bind an epitope of CD19 encoded by exon4 of the CD19 gene.

CD19ALAb

The present inventors have developed a new anti-CD19 CAR which hasimproved properties compared to a known anti-CD19 CAR which comprisesthe binder fmc63 (see Examples 2 and 3). The antigen binding domain ofthe CAR is based on the CD19 binder CDI9ALAb, which has the CDRs andVH/VL regions identified below.

The present invention therefore also provides a CAR which comprises aCD19-binding domain which comprises a) a heavy chain variable region(VH) having complementarity determining regions (CDRs) with thefollowing sequences:

(SEQ ID No. 15) CDR1-SYWMN; (SEQ ID No. 16) CDR2-QIWPGDGDTNYNGKFK(SEQ ID No. 17) CDR3-RETTTVGRYYYAMDY;and

b) a light chain variable region (VL) having CDRs with the followingsequences:

(SEQ ID No. 18) CDR1-KASQSVDYDGDSYLN; (SEQ ID No. 19) CDR2-DASNLVS(SEQ ID No. 20) CDR3-QQSTEDPWT.

It may be possible to introduce one or more mutations (substitutions,additions or deletions) into the or each CDR without negativelyaffecting CD19-binding activity. Each CDR may, for example, have one,two or three amino acid mutations.

The CAR of the present invention may comprise one of the following aminoacid sequences:

(Murine CD19ALAb scFv sequence)  SEQ ID No. 21QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK(Humanised CD19ALAb scFv sequence-Heavy 19,  Kappa 16) SEQ ID No. 22QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGTLVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR(Humanised CD19ALAb scFv sequence-Heavy 19, Kappa 7) SEQ ID No. 39QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGTLVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIKR

The scFv may be in a VH-VL orientation (as shown in SEQ ID No.s 21, 22and 39) or a VL-VH orientation.

The CAR of the present invention may comprise one of the following VHsequences:

(Murine CD19ALAb VH sequence) SEQ ID No. 23QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (Humanised CD19ALAb VH sequence) SEQ ID No. 24QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGTLVTVSS

The CAR of the present invention may comprise one of the following VLsequences:

(Murine CD19ALAb VL sequence) SEQ ID No. 25DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPW TFGGGTKLEIK(Humanised CD19ALAb VL sequence, Kappa 16) SEQ ID No. 26DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPW TFGQGTKVEIKR(Humanised CD19ALAb VL sequence, Kappa 7) SEQ ID No. 40DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPW TFGQGTKVEIKR

The CAR of the invention may comprise a variant of the sequence shown asSEQ ID No. 21, 22, 23, 24, 25, 26, 39 or 40 having at least 80, 85, 90,95, 98 or 99% sequence identity, provided that the variant sequenceretain the capacity to bind CD19 (when in conjunction with acomplementary VL or VH domain, if appropriate).

The percentage identity between two polypeptide sequences may be readilydetermined by programs such as BLAST which is freely available athttp://blast.ncbi.nlm.nih.gov.

CD22

The human CD22 antigen is a molecule belonging to the SIGLEC family oflectins. It is found on the surface of mature B cells and on someimmature B cells. Generally speaking, CD22 is a regulatory molecule thatprevents the overactivation of the immune system and the development ofautoimmune diseases.

CD22 is a sugar binding transmembrane protein, which specifically bindssialic acid with an immunoglobulin (Ig) domain located at itsN-terminus. The presence of Ig domains makes CD22 a member of theimmunoglobulin superfamily. CD22 functions as an inhibitory receptor forB cell receptor (BCR) signaling.

CD22 is a molecule of the IgSF which may exist in two isoforms, one withseven domains and an intra-cytoplasmic tail comprising of three ITIMs(immune receptor tyrosine-based inhibitory motifs) and an ITAM; and asplicing variant which instead comprises of five extracellular domainsand an intra-cytoplasmic tail carrying one ITIM. CD22 is thought to bean inhibitory receptor involved in the control of B-cell responses toantigen. Like CD19, CD22 is widely considered to be a pan-B antigen,although expression on some non-lymphoid tissue has been described.Targeting of CD22 with therapeutic monoclonal antibodies andimmunoconjugates has entered clinical testing.

Examples of anti-CD22 CARs are described by Haso et al. (Blood; 2013;121(7)). Specifically, anti-CD22 CARs with antigen-binding domainsderived from m971, HA22 and BL22 scFvs are described.

The antigen-binding domain of the anti-CD22 CAR may bind CD22 with aK_(D) in the range 30-50 nM, for example 30-40 nM. The K_(D) may beabout 32 nM.

CD-22 has seven extracellular IgG-like domains, which are commonlyidentified as Ig domain 1 to Ig domain 7, with Ig domain 7 being mostproximal to the B cell membrane and Ig domain 7 being the most distalfrom the Ig cell membrane (see Haso et al 2013 as above FIG. 2B).

The positions of the Ig domains in terms of the amino acid sequence ofCD22 (http://www.uniprot.org/uniprot/P20273) are summarised in thefollowing table:

Ig domain Amino acids 1  20-138 2 143-235 3 242-326 4 331-416 5 419-5006 505-582 7 593-676

The antigen-binding domain of the second CAR may bind to amembrane-distal epitope on CD22. The antigen-binding domain of thesecond CAR may bind to an epitope on Ig domain 1, 2, 3 or 4 of CD22, forexample on Ig domain 3 of CD22. The antigen-binding domain of the secondCAR may bind to an epitope located between amino acids 20-416 of CD22,for example between amino acids 242-326 of CD22.

The anti-CD22 antibodies HA22 and BL22 (Haso et al 2013 as above) andCD22ALAb, described below, bind to an epitope on Ig domain 3 of CD22.

The antigen binding domain of the second CAR may not bind to amembrane-proximal epitope on CD22. The antigen-binding domain of thesecond CAR may not bind to an epitope on Ig domain 5, 6 or 7 of CD22.The antigen-binding domain of the second CAR may not bind to an epitopelocated between amino acids 419-676 of CD22, such as between 505-676 ofCD22.

CD22ALAb

The present inventors have developed a new anti-CD22 CAR which hasimproved properties compared to a known anti-CD22 CAR which comprisesthe binder m971 (see Examples 2 and 3 and Haso et al (2013) as above).The antigen binding domain of the CAR is based on the CD22 binderCD22ALAb, which has the CDRs and VH/VL regions identified below.

The present invention therefore also provides a CAR which comprises aCD22-binding domain which comprises

a) a heavy chain variable region (VH) having complementarity determiningregions (CDRs) with the following sequences:

CDR1 (SEQ ID No. 27) NYWIN; CDR2 (SEQ ID No. 28) NIYPSDSFTNYNQKFKD CDR3(SEQ ID No. 29) DTQERSWYFDV;and

b) a light chain variable region (VL) having CDRs with the followingsequences:

CDR1 (SEQ ID No. 30) RSSQSLVHSNGNTYLH; CDR2 (SEQ ID No. 31) KVSNRFS CDR3(SEQ ID No. 32) SQSTHVPWT.

It may be possible to introduce one or more mutations (substitutions,additions or deletions) into the or each CDR without negativelyaffecting CD22-binding activity. Each CDR may, for example, have one,two or three amino acid mutations.

The CAR of the present invention may comprise one of the following aminoacid sequences:

(Murine CD22ALAb scFv sequence) SEQ ID No. 33QVQLQQPGAELVRPGASVKLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIYPSDSFTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRDTQERSWYFDVWGAGTTVTVSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPWTFGGGTKLEIK (Humanised CD22ALAb scFv sequence)SEQ ID No. 34 EVQLVESGAEVKKPGSSVKVSCKASGYTFTNYWINWVRQAPGQGLEWIGNIYPSDSFTNYNQKFKDRATLTVDKSTSTAYLELRNLRSDDTAVYYCTRDTQERSWYFDVWGQGTLVTVSSDIVMTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSNRFSGVPARFSGSGSGVEFTLTISSLQSEDFAVYYCSQSTHVPWTFGQGTRLEIK

The scFv may be in a VH-VL orientation (as shown in SEQ ID Nos 33 and34) or a VL-VH orientation.

The CAR of the present invention may comprise one of the following VHsequences:

(Murine CD22ALAb VH sequence) SEQ ID No. 35QVQLQQPGAELVRPGASVKLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIYPSDSFTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRDT QERSWYFDVWGAGTTVTVSS(Humanised CD22ALAb VH sequence) SEQ ID No. 36EVQLVESGAEVKKPGSSVKVSCKASGYTFTNYWINWVRQAPGQGLEWIGNIYPSDSFTNYNQKFKDRATLTVDKSTSTAYLELRNLRSDDTAVYYCTRDT QERSWYFDVWGQGTLVTVSS

The CAR of the present invention may comprise one of the following VLsequences:

(Murine CD22ALAb VL sequence) SEQ ID No. 37DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVP WTFGGGTKLEIK(Humanised CD22ALAb VL sequence) SEQ ID No. 38DIVMTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSNRFSGVPARFSGSGSGVEFTLTISSLQSEDFAVYYCSQSTHVP WTFGQGTRLEIK

The CAR of the invention may comprise a variant of the sequence shown asSEQ ID No. 33, 34, 35, 36, 37 or 38 having at least 80, 85, 90, 95, 98or 99% sequence identity, provided that the variant sequence retain thecapacity to bind CD22 (when in conjunction with a complementary VL or VHdomain, if appropriate).

B-Cell Antigen Expression During B-Cell Ontogeny and Subsequent Tumours

CD19 is widely considered a pan-B antigen, although very occasionally,it may display some lineage infidelity. The CD19 molecule comprises oftwo extracellular IgSF domains separated by a smaller domain and a longintracytoplasmic tail, nearly as big as the extracellular portion of themolecule, carrying one ITAM. CD19 is a key molecule in the developmentand activation of B-cells. CD22 is a molecule of the IgSF which mayexist in two isoforms, one with seven domains and an intra-cytoplasmictail comprising of three ITIMs (immune receptor tyrosine-basedinhibitory motifs) and an ITAM; and a splicing variant which insteadcomprises of five extracellular domains and an intra-cytoplasmic tailcarrying one ITIM. CD22 is thought to be an inhibitory receptor involvedin the control of B-cell responses to antigen. Like CD19, CD22 is widelyconsidered to be a pan-B antigen, although expression on somenon-lymphoid tissue has been described (Wen et al. (2012) J. Immunol.Baltim. Md. 1950 188, 1075-1082). Targeting of CD22 with therapeuticmonoclonal antibodies and immunoconjugates has entered clinical testing.Generation of CD22 specific CARs have been described (Naso et al, 2013,Blood: Volume 121; 7: 1165-74, and James et al 2008, Journal ofimmunology, Volume 180; Issue 10; Pages 7028-38).

Detailed immunophentyping studies of B-cell leukaemias shows that whilesurface CD19 is always present, surface CD22 is almost always present.For instance, Raponi et al (2011, as above) studied the surface antigenphenotype of 427 cases of B-ALL and found CD22 present in 341 of casesstudied.

The eventuality of CD19 down-regulation after CAR19 targeting describedabove may be explained by the Goldie-Coldman hypothesis. TheGoldie-Coldman hypothesis predicts that tumor cells mutate to aresistant phenotype at a rate dependent on their intrinsic geneticinstability and that the probability that a cancer would containresistant clones depends on the mutation rate and the size of the tumor.While it may be difficult for cancer cells to become intrinsicallyresistant to the direct killing of cytotoxic T-cells, antigen lossremains possible. Indeed this phenomenon has been reported before withtargeting melanoma antigens and EBV-driven lymphomas. According toGoldie-Coldman hypothesis, the best chance of cure would be tosimultaneously attack non-cross resistant targets. Given that CD22 isexpressed on nearly all cases of B-ALL, simultaneous CAR targeting ofCD19 along with CD22 may reduce the emergence of resistant CD19 negativeclones.

Antigen Binding Domain

The antigen binding domain is the portion of the CAR which recognizesantigen. Numerous antigen-binding domains are known in the art,including those based on the antigen binding site of an antibody,antibody mimetics, and T-cell receptors. For example, theantigen-binding domain may comprise: a single-chain variable fragment(scFv) derived from a monoclonal antibody; a natural ligand of thetarget antigen; a peptide with sufficient affinity for the target; asingle domain antibody; an artificial single binder such as a Darpin(designed ankyrin repeat protein); or a single-chain derived from aT-cell receptor.

The antigen binding domain of the CAR which binds to CD19 may be anydomain which is capable of binding CD19. For example, the antigenbinding domain may comprise a CD19 binder as described in Table 1.

The antigen binding domain of the CAR which binds to CD19 may comprise asequence derived from one of the CD19 binders shown in Table 2.

TABLE 2 Binder References HD63 Pezzutto (Pezzutto, A. et al. J. Immunol.Baltim. Md 1950 138, 2793-2799 (1987) 4g7 Meeker et al (Meeker, T. C. etal. Hybridoma 3, 305-320 (1984) Fmc63 Nicholson et al (Nicholson, I. C.et al. Mol. Immunol. 34, 1157-1165 (1997) B43 Bejcek et al (Bejcek, B.E. et al. Cancer Res. 55, 2346-2351 (1995) SJ25C1 Bejcek et al (1995, asabove) BLY3 Bejcek et al (1995, as above) B4, or re-surfaced, Roguska etal (Roguska, M. A. et al. Protein or humanized B4 Eng. 9, 895-904 (1996)HB12b, optimized Kansas et al (Kansas, G. S. & Tedder, T. F. J. andhumanized Immunol. Baltim. Md 1950 147, 4094-4102 (1991); Yazawa et al(Yazawa et al Proc. Natl. Acad. Sci. U.S.A. 102, 15178-15183 (2005);Herbst et al (Herbst, R. et al. J. Pharmacol. Exp. Ther. 335, 213-222(2010)

The antigen binding domain of the CAR which binds to CD22 may be anydomain which is capable of binding CD22. For example, the antigenbinding domain may comprise a CD22 binder as described in Table 3.

TABLE 3 Binder References M5/44 or humanized M5/44 John et al (J.Immunol. Baltim. Md 1950 170, 3534-3543 (2003); and DiJoseph et al(Cancer Immunol. Immunother. CII 54, 11-24 (2005) M6/13 DiJoseph et al(as above) HD39 Dorken et al (J. Immunol. Baltim. Md 1950 135, 4470-4479(1986) HD239 Dorken et al (as above) HD6 Pezzutto et al (J. Immunol.Baltim. Md 1950 138, 98-103 (1987) RFB-4, or Campana et al (J. Immunol.Baltim. Md 1950 134,1524-1530 humanized RFB-4, or (1985); Krauss et al(Protein Eng. 16, 753-759 (2003), affinity matured Kreitman et al (J.Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 30, 1822-1828 (2012)) To15Mason et al (Blood 69, 836-840 (1987)) 4KB128 Mason et al (as above)S-HCL1 Schwarting et al (Blood 65, 974-983 (1985)) mLL2 (EPB-2), or Shihet al (Int. J. Cancer J. Int. Cancer 56, 538-545 (1994)), humanizedmLL2- Leonard et al (J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 21,hLL2 3051-3059 (2003)) M971 Xiao et al (mAbs 1, 297-303 (2009)) BC-8Engel et al (J. Exp. Med. 181, 1581-1586 (1995)) HB22-12 Engel et al (asabove)

Spacer Domain

CARs comprise a spacer sequence to connect the antigen-binding domainwith the transmembrane domain and spatially separate the antigen-bindingdomain from the endodomain. A flexible spacer allows the antigen-bindingdomain to orient in different directions to facilitate binding.

In the cell of the present invention, the first and second CARs maycomprise different spacer molecules. For example, the spacer sequencemay, for example, comprise an IgG1 Fc region, an IgG1 hinge or a humanCD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprisean alternative linker sequence which has similar length and/or domainspacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 stalk. Ahuman IgG1 spacer may be altered to remove Fc binding motifs.

The spacer for the anti-CD19 CAR may comprise a CD8 stalk spacer, or aspacer having a length equivalent to a CD8 stalk spacer. The spacer forthe anti-CD19 CAR may have at least 30 amino acids or at least 40 aminoacids. It may have between 35-55 amino acids, for example between 40-50amino acids. It may have about 46 amino acids.

The spacer for the anti-CD22 CAR may comprise an IgG1 hinge spacer, or aspacer having a length equivalent to an IgG1 hinge spacer. The spacerfor the anti-CD22 CAR may have fewer than 30 amino acids or fewer than25 amino acids. It may have between 15-25 amino acids, for examplebetween 18-22 amino acids. It may have about 20 amino acids.

Examples of amino acid sequences for these spacers are given below:

(hinge-CH2CH3 of human IgG1) SEQ ID No. 4AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD (human CD8 stalk): SEQ ID No. 5TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (human IgG1 hinge):SEQ ID No. 6 AEPKSPDKTHTCPPCPKDPK (CD2 ectodomain) SEQ ID No. 7KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD (CD34 ectodomain) SEQ ID No. 8SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVA SHQSYSQKT

Since CARs are typically homodimers (see FIG. 1a ), cross-pairing mayresult in a heterodimeric chimeric antigen receptor. This is undesirablefor various reasons, for example: (1) the epitope may not be at the same“level” on the target cell so that a cross-paired CAR may only be ableto bind to one antigen; (2) the VH and VL from the two different scFvcould swap over and either fail to recognize target or worse recognizean unexpected and unpredicted antigen. The spacer of the first CAR maybe sufficiently different from the spacer of the second CAR in order toavoid cross-pairing. The amino acid sequence of the first spacer mayshare less that 50%, 40%, 30% or 20% identity at the amino acid levelwith the second spacer.

Transmembrane Domain

The transmembrane domain is the sequence of the CAR that spans themembrane.

A transmembrane domain may be any protein structure which isthermodynamically stable in a membrane. This is typically an alpha helixcomprising of several hydrophobic residues. The transmembrane domain ofany transmembrane protein can be used to supply the transmembraneportion of the invention. The presence and span of a transmembranedomain of a protein can be determined by those skilled in the art usingthe TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/).Further, given that the transmembrane domain of a protein is arelatively simple structure, i.e a polypeptide sequence predicted toform a hydrophobic alpha helix of sufficient length to span themembrane, an artificially designed TM domain may also be used (U.S. Pat.No. 7,052,906 B1 describes synthetic transmembrane components).

The transmembrane domain may be derived from CD28, which gives goodreceptor stability.

The transmembrane domain may be derived from human Tyrp-1. The tyrp-1transmembrane sequence is shown as SEQ ID No. 45.

SEQ ID No. 45 IIAIAVVGALLLVALIFGTASYLI

ACTIVATING ENDODOMAIN

The endodomain is the signal-transmission portion of the CAR. Afterantigen recognition, receptors cluster, native CD45 and CD148 areexcluded from the synapse and a signal is transmitted to the cell. Themost commonly used endodomain component is that of CD3-zeta whichcontains 3 ITAMs. This transmits an activation signal to the T cellafter antigen is bound. CD3-zeta may not provide a fully competentactivation signal and additional co-stimulatory signaling may be needed.For example, chimeric CD28 and OX40 can be used with CD3-Zeta totransmit a proliferative/survival signal, or all three can be usedtogether.

The cell of the present invention comprises two CARs, each with anendodomain.

The endodomain of the first CAR and the endodomain of the second CAR maycomprise:

-   -   (i) an ITAM-containing endodomain, such as the endodomain from        CD3 zeta; and/or    -   (ii) a co-stimulatory domain, such as the endodomain from CD28;        and/or    -   (iii) a domain which transmits a survival signal, for example a        TNF receptor family endodomain such as OX-40 or 4-1BB.

In one arrangement the co-stimulatory and survival signal-producingdomains are “shared” between the two (or more) CARs in an OR gate. Forexample, where an OR gate has two CARs, CAR A and CAR B, CAR A maycomprise a co-stimulatory domain (e.g. CD28 endodomain) and CARB maycomprise a TNF receptor family endodomain, such as OX-40 or 4-1BB.

An endodomain which contains an ITAM motif can act as an activationendodomain in this invention. Several proteins are known to containendodomains with one or more ITAM motifs. Examples of such proteinsinclude the CD3 epsilon chain, the CD3 gamma chain and the CD3 deltachain to name a few. The ITAM motif can be easily recognized as atyrosine separated from a leucine or isoleucine by any two other aminoacids, giving the signature YxxL/I. Typically, but not always, two ofthese motifs are separated by between 6 and 8 amino acids in the tail ofthe molecule (YxxL/Ix(6-8)YxxL/1). Hence, one skilled in the art canreadily find existing proteins which contain one or more ITAM totransmit an activation signal. Further, given the motif is simple and acomplex secondary structure is not required, one skilled in the art candesign polypeptides containing artificial ITAMs to transmit anactivation signal (see WO 2000/063372, which relates to syntheticsignalling molecules).

The transmembrane and intracellular T-cell signalling domain(endodomain) of a CAR with an activating endodomain may comprise thesequence shown as SEQ ID No. 9, 10 or 11 or a variant thereof having atleast 80% sequence identity.

comprising CD28 transmembrane domain and CD3 Z  endodomain SEQ ID No. 9FWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRcomprising CD28 transmembrane domain and CD28 and  CD3 Zeta endodomainsSEQ ID No. 10 FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRcomprising CD28 transmembrane domain and CD28, OX40and CD3 Zeta endodomains SEQ ID No. 11FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR

A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99%sequence identity to SEQ ID No. 9, 10 or 11, provided that the sequenceprovides an effective trans-membrane domain and an effectiveintracellular T cell signaling domain.

“Split” or Gate Endodomains

The present invention provides an OR gate in which theco-stimulatory/survival signal domains are “split” between the two CARs.

In this respect, the present invention provides a cell whichco-expresses a first chimeric antigen receptor (CAR) and second CAR atthe cell surface, each CAR comprising an intracellular signallingdomain, wherein the intracellular signalling domain of the first CARcomprises a co-stimulatory domain; and the intracellular signallingdomain of the second CAR comprises a TNF receptor family endodomain.

The first and second CARs may bind to different antigens. For example,the first CAR may bind CD19 and the second CAR may bind CD22;alternatively the first CAR may bind CD22 and the second CAR may bindCD19.

The intracellular signalling domain of the first CAR comprises aco-stimulatory domain and does not comprise a domain which transmitssurvival signals (such as a TNF receptor family endodomain). Theintracellular signalling domain of the second CAR comprises a TNFreceptor family endodomain and does not comprise a co-stimulatory domain(such as CD28 endodomain).

The co-stimulatory domain may be a CD28 co-stimulatory domain. The CD28co-stimulatory domain may have the sequence shown as SEQ ID No. 41.

(CD28 co-stimulatory endodomain) SEQ ID No. 41SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

The CAR of the invention may comprise a variant of the sequence shown asSEQ ID No. 41 having at least 80, 85, 90, 95, 98 or 99% sequenceidentity, provided that the variant sequence retains the capacity toco-stimulate T cells upon antigen recognition, i.e. provide signal 2 toT cells.

The TNF receptor family endodomain may be an OX40 or 4-1BB endodomain.The OX40 endodomain may have the sequence shown as SEQ ID No. 42. The4-1BB endodomain may have the sequence shown as SEQ ID No. 43.

(OX40 endodomain) SEQ ID No. 42 RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI(4-1BB endodomain) SEQ ID No. 43KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

The CAR of the invention may comprise a variant of the sequence shown asSEQ ID No. 42 or 43 having at least 80, 85, 90, 95, 98 or 99% sequenceidentity, provided that the variant sequence retains the capacity totransmit a survival signal to T cells upon antigen recognition.

The intracellular signalling domain of the first and/or the second CARmay also comprise an ITAM-containing domain, such as a CD3 zeta domain.The CD3 zeta domain may have the sequence shown as SEQ ID No. 44.

(CD3zeta endodomain) SEQ ID No. 44RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

The CAR of the invention may comprise a variant of the sequence shown asSEQ ID No. 44 having at least 80, 85, 90, 95, 98 or 99% sequenceidentity, provided that the variant sequence retains the capacity toinduce T-cell signalling upon antigen recognition, i.e. provide signal 1to T cells.

The first CAR may have the structure:

-   -   AgB1-spacerl-TM1-costim-ITAM

in which:

AgB1 is the antigen-binding domain of the first CAR;

spacer 1 is the spacer of the first CAR;

TM1 is the transmembrane domain of the first CAR;

costim is a co-stimulatory domain; and

ITAM is an ITAM-containing endodomain.

“Costim” may be a CD28 co-stimulatory domain.

“ITAM” may be a CD3 zeta endodomain.

The second CAR may have the structure:

-   -   AgB2-spacer2-TM2-TNF-ITAM

in which:

AgB2 is the antigen-binding domain of the second CAR;

spacer 2 is the spacer of the second CAR;

TM2 is the transmembrane domain of the second CAR;

TNF is a TNF receptor endodomain; and

ITAM is an ITAM-containing endodomain.

“TNF” may be a TNF receptor endodomain such as the OX40 or 4-1BBendodomains.

There is also provided a nucleic acid sequence encoding both the firstand second chimeric antigen receptors (CARs) with “split” endodomains;and a kit comprising two nucleic acids one encoding a first CAR and oneencoding a second CAR comprising split endodomains as defined above.

Co-Expression Site

The second aspect of the invention relates to a nucleic acid whichencodes the first and second CARs.

The nucleic acid may produce a polypeptide which comprises the two CARmolecules joined by a cleavage site. The cleavage site may beself-cleaving, such that when the polypeptide is produced, it isimmediately cleaved into the first and second CARs without the need forany external cleavage activity.

Various self-cleaving sites are known, including the Foot-and-Mouthdisease virus (FMDV) 2A peptide and similar sequence (Donnelly et al,Journal of General Virology (2001), 82, 1027-1041), for instance likethe 2A-like sequence from Thosea asigna virus which has the sequenceshown as SEQ ID No. 12:

SEQ ID No. 12 RAEGRGSLLTCGDVEENPGP.

The co-expressing sequence may be an internal ribosome entry sequence(IRES). The co-expressing sequence may be an internal promoter

Cell

The present invention relates to a cell which co-expresses a first CARand a second CAR at the cell surface, wherein one CAR binds CD19 and theother CAR binds CD22.

The cell may be any eukaryotic cell capable of expressing a CAR at thecell surface, such as an immunological cell.

In particular the cell may be an immune effector cell such as a T cellor a natural killer (NK) cell.

T cells or T lymphocytes are a type of lymphocyte that play a centralrole in cell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR) on the cell surface. There arevarious types of T cell, as summarised below.

Helper T helper cells (TH cells) assist other white blood cells inimmunologic processes, including maturation of B cells into plasma cellsand memory B cells, and activation of cytotoxic T cells and macrophages.TH cells express CD4 on their surface. TH cells become activated whenthey are presented with peptide antigens by MHC class II molecules onthe surface of antigen presenting cells (APCs). These cells candifferentiate into one of several subtypes, including TH1, TH2, TH3,TH17, Th9, or TFH, which secrete different cytokines to facilitatedifferent types of immune responses.

Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells andtumor cells, and are also implicated in transplant rejection. CTLsexpress the CD8 at their surface. These cells recognize their targets bybinding to antigen associated with MHC class I, which is present on thesurface of all nucleated cells. Through IL-10, adenosine and othermolecules secreted by regulatory T cells, the CD8+ cells can beinactivated to an anergic state, which prevent autoimmune diseases suchas experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise three subtypes: central memory T cells (TCMcells) and two types of effector memory T cells (TEM cells and TEMRAcells). Memory cells may be either CD4+or CD8+. Memory T cells typicallyexpress the cell surface protein CD45RO.

Regulatory T cells (Treg cells), formerly known as suppressor T cells,are crucial for the maintenance of immunological tolerance. Their majorrole is to shut down T cell-mediated immunity toward the end of animmune reaction and to suppress auto-reactive T cells that escaped theprocess of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described—naturallyoccurring Treg cells and adaptive Treg cells.

Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Tregcells) arise in the thymus and have been linked to interactions betweendeveloping T cells with both myeloid (CD11c+) and plasmacytoid (CD123+)dendritic cells that have been activated with TSLP.

Naturally occurring Treg cells can be distinguished from other T cellsby the presence of an intracellular molecule called FoxP3. Mutations ofthe FOXP3 gene can prevent regulatory T cell development, causing thefatal autoimmune disease IPEX.

Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originateduring a normal immune response.

The T cell of the invention may be any of the T cell types mentionedabove, in particular a CTL.

Natural killer (NK) cells are a type of cytolytic cell which forms partof the innate immune system. NK cells provide rapid responses to innatesignals from virally infected cells in an MHC independent manner

NK cells (belonging to the group of innate lymphoid cells) are definedas large granular lymphocytes (LGL) and constitute the third kind ofcells differentiated from the common lymphoid progenitor generating Band T lymphocytes. NK cells are known to differentiate and mature in thebone marrow, lymph node, spleen, tonsils and thymus where they thenenter into the circulation.

The CAR cells of the invention may be any of the cell types mentionedabove.

CAR-expressing cells, such as CAR-expressing T or NK cells may either becreated ex vivo either from a patient's own peripheral blood (1stparty), or in the setting of a haematopoietic stem cell transplant fromdonor peripheral blood (2nd party), or peripheral blood from anunconnected donor (3rd party).

The present invention also provide a cell composition comprising CARexpressing T cells and/or CAR expressing NK cells according to thepresent invention. The cell composition may be made by transducing ablood-sample ex vivo with a nucleic acid according to the presentinvention.

Alternatively, CAR-expressing cells may be derived from ex vivodifferentiation of inducible progenitor cells or embryonic progenitorcells to the relevant cell type, such as T cells. Alternatively, animmortalized cell line such as a T-cell line which retains its lyticfunction and could act as a therapeutic may be used.

In all these embodiments, CAR cells are generated by introducing DNA orRNA coding for the CARs by one of many means including transduction witha viral vector, transfection with DNA or RNA.

A CAR T cell of the invention may be an ex vivo T cell from a subject.The T cell may be from a peripheral blood mononuclear cell (PBMC)sample. T cells may be activated and/or expanded prior to beingtransduced with CAR-encoding nucleic acid, for example by treatment withan anti-CD3 monoclonal antibody.

A CAR T cell of the invention may be made by:

-   -   (i) isolation of a T cell-containing sample from a subject or        other sources listed above; and    -   (ii) transduction or transfection of the T cells with one or        more nucleic acid sequence(s) encoding the first and second CAR.

The T cells may then by purified, for example, selected on the basis ofco-expression of the first and second CAR.

Nucleic Acid Sequences

The second aspect of the invention relates to one or more nucleic acidsequence(s) which codes for a first CAR and a second CAR as defined inthe first aspect of the invention.

The nucleic acid sequence may be, for example, an RNA, a DNA or a cDNAsequence.

The nucleic acid sequence may encode one chimeric antigen receptor (CAR)which binds to CD19 and another CAR which binds to CD22.

The nucleic acid sequence may have the following structure:

-   -   AgB1-spacer/-TM1-coexpr-AbB2-spacer2-TM2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain of afirst CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of a first CAR;

TM1 is a a nucleic acid sequence encoding the transmembrane domain of afirst CAR;

coexpr is a nucleic acid sequence enabling co-expression of both CARs

AgB2 is a nucleic acid sequence encoding the antigen-binding domain of asecond CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of a second CAR;

TM2 is a a nucleic acid sequence encoding the transmembrane domain of asecond CAR;

which nucleic acid sequence, when expressed in a T cell, encodes apolypeptide which is cleaved at the cleavage site such that the firstand second CARs are co-expressed at the cell surface.

The first CAR may bind CD19 and the second CAR may bind CD22.Alternatively the first CAR may bind CD22 and the second CAR may bindCD19.

Alternative codons may be used in regions of sequence encoding the sameor similar amino acid sequences, in order to avoid homologousrecombination.

Due to the degeneracy of the genetic code, it is possible to usealternative codons which encode the same amino acid sequence. Forexample, the codons “ccg” and “cca” both encode the amino acid proline,so using “ccg” may be exchanged for “cca” without affecting the aminoacid in this position in the sequence of the translated protein.

The alternative RNA codons which may be used to encode each amino acidare summarised in Table 3.

TABLE 3 U C A G U

C

A

G

Alternative codons may be used in the portions of nucleic acid sequencewhich encode the spacer of the first CAR and the spacer of the secondCAR, especially if the same or similar spacers are used in the first andsecond CARs. FIG. 4 shows two sequences encoding the spacerHCH2CH3-hinge, in one of which alternative codons have been used.

Alternative codons may be used in the portions of nucleic acid sequencewhich encode the transmembrane domain of the first CAR and thetransmembrane of the second CAR, especially if the same or similartransmembrane domains are used in the first and second CARs. FIG. 4shows two sequences encoding the CD28 transmembrane domain, in one ofwhich alternative codons have been used.

Alternative codons may be used in the portions of nucleic acid sequencewhich encode all or part of the endodomain of the first CAR and all orpart of the endodomain of the second

CAR. Alternative codons may be used in the CD3 zeta endodomain. FIG. 4shows two sequences encoding the CD3 zeta endodomain, in one of whichalternative codons have been used.

Alternative codons may be used in one or more co-stimulatory domains,such as the CD28 endodomain.

Alternative codons may be used in one or more domains which transmitsurvival signals, such as OX40 and 41BB endodomains.

Alternative codons may be used in the portions of nucleic acid sequenceencoding a CD3zeta endodomain and/or the portions of nucleic acidsequence encoding one or more costimulatory domain(s) and/or theportions of nucleic acid sequence encoding one or more domain(s) whichtransmit survival signals.

Vector

The present invention also provides a vector, or kit of vectors whichcomprises one or more CAR-encoding nucleic acid sequence(s). Such avector may be used to introduce the nucleic acid sequence(s) into a hostcell so that it expresses the first and second CARs.

The vector may, for example, be a plasmid or a viral vector, such as aretroviral vector or a lentiviral vector, or a transposon based vectoror synthetic mRNA.

The vector may be capable of transfecting or transducing a T cell.

Pharmaceutical Composition

The present invention also relates to a pharmaceutical compositioncontaining a plurality of CAR-expressing cells, such as T cells or NKcells according to the first aspect of the invention. The pharmaceuticalcomposition may additionally comprise a pharmaceutically acceptablecarrier, diluent or excipient. The pharmaceutical composition mayoptionally comprise one or more further pharmaceutically activepolypeptides and/or compounds. Such a formulation may, for example, bein a form suitable for intravenous infusion.

Method of Treatment

The cells of the present invention are capable of killing cancer cells,such as B-cell lymphoma cells. CAR-expressing cells, such as T cells,may either be created ex vivo either from a patient's own peripheralblood (1st party), or in the setting of a haematopoietic stem celltransplant from donor peripheral blood (2nd party), or peripheral bloodfrom an unconnected donor (3rd party). Alternatively, CAR T-cells may bederived from ex-vivo differentiation of inducible progenitor cells orembryonic progenitor cells to T-cells. In these instances, CAR T-cellsare generated by introducing DNA or RNA coding for the CAR by one ofmany means including transduction with a viral vector, transfection withDNA or RNA.

The cells of the present invention may be capable of killing targetcells, such as cancer cells. The target cell is recognisable byexpression of CD19 or CD22.

TABLE 4 expression of lymphoid antigens on lymphoid leukaemias CD19 CD22CD10 CD7 CD5 CD3 cIg μ sig μ Early pre-B 100 >95 95 5 0 0 0 0 Pre-B 100100 >95 0 0 0 100 0 Transitional 100 100 50 0 0 0 100 0 pre-B B 100 10050 0 0 0 >95 >95 T <5 0 0 100 95 100 0 0

Taken from Campana et al. (Immunophenotyping of leukemia. J. lmmunol.Methods 243, 59-75 (2000)). clg μ—cytoplasic Immunoglobulin heavy chain;slg μ—surface Immunoglobulin heavy chain.

The expression of commonly studied lymphoid antigens on different typesof B-cell leukaemias closely mirrors that of B-cell ontogeny (see FIG.2).

The T cells of the present invention may be used to treat cancer, inparticular B-cell malignancies.

Examples of cancers which express CD19 or CD22 are B-cell lymphomas,including Hodgkin's lymphoma and non-Hodgkins lymphoma; and B-cellleukaemias.

For example the B-cell lymphoma may be Diffuse large B cell lymphoma(DLBCL), Follicular lymphoma, Marginal zone lymphoma (MZL) orMucosa-Associated Lymphatic Tissue lymphoma (MALT), Small celllymphocytic lymphoma (overlaps with Chronic lymphocytic leukemia),Mantle cell lymphoma (MCL), Burkitt lymphoma, Primary mediastinal(thymic) large B-cell lymphoma, Lymphoplasmacytic lymphoma (may manifestas Waldenström macroglobulinemia), Nodal marginal zone B cell lymphoma(NMZL), Splenic marginal zone lymphoma (SMZL), Intravascular largeB-cell lymphoma, Primary effusion lymphoma, Lymphomatoid granulomatosis,T cell/histiocyte-rich large B-cell lymphoma or Primary central nervoussystem lymphoma.

The B-cell leukaemia may be acute lymphoblastic leukaemia, B-cellchronic lymphocytic leukaemia, B-cell prolymphocytic leukaemia,precursor B lymphoblastic leukaemia or hairy cell leukaemia.

The B-cell leukaemia may be acute lymphoblastic leukaemia.

Treatment with the T cells of the invention may help prevent the escapeor release of tumour cells which often occurs with standard approaches.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1 Proof-of-Concept of a CD19/CD22 Logical ‘OR’ Gate

A CD19 ‘OR’ CD22 CAR gate was constructed by co-expression of a CD19 anda CD22 CAR in the same vector. The anti-CD19 binder was a scFv derivedfrom the re-surfaced B4 antibody (Roguska et al. (1996) Protein Eng. 9,895-904), and the anti-CD22 binder was a scFv derived from the humanizedRFB4 antibody. A human IgG1 hinge-CH2-CH3 spacer was used for both CARs,the coding sequence of which was codon-wobbled to avoid homologousrecombination by the integrating vector. The TM domain in both CARs wasderived from that of CD28, and both CAR endodomains comprised ofCD3-Zeta. Once again, these homologous sequences were codon-wobbled.Co-expression was achieved by cloning the two CARs in frame separated bya FMD-2A peptide. The nucleic acid and amino acid sequence of theCD19/CD22 ‘OR’ gate construct are shown as SEQ ID NOs: 13 and 14;respectively.

SEQ ID NO: 13 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCATACCCCTACGACGTGCCCGACTACGCCAGCCTGAGCGGAGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTGAAGGTGTCCTGTAAGGCCAGCGGCTACACCTTCACCAGCAACTGGATGCACTGGGTGAGGCAGGCCCCTGGACAGGGACTGGAGTGGATGGGCGAGATCGACCCCAGCGACAGCTACACCAACTACAACCAGAAGTTCAAGGGCCGGGTGACCATCACCGTGGATAAGAGCGCCAGCACCGCCTACATGGAGCTGTCCAGCCTGAGAAGCGAGGATACCGCCGTGTACTACTGTGCCAGAGGCAGCAACCCCTACTACTACGCTATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCAGCGGCGGAGGAGGAAGCGGAGGGGGCGGATCTGGCGGCGGAGGGAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGTAGCGCCAGCAGCGGCGTGAATTACATGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGAAGATGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCCGCCAGATTCAGCGGCAGCGGCTCCGGCACCAGCTACAGCCTGACCATCAGCAGCCTGGAGCCTGAGGATTTCGCCGTGTATTATTGCCACCAGAGGGGCAGCTACACCTTTGGCGGCGGAACAAAGCTGGAGATCAAGCGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCAGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTATCTATGACATGTCTTGGGTCCGCCAGGTTCCGGGGAAGGGGCTGGAGTGGGTCTCATATATTAGTAGTGGTGGTGGTACCACCTATTACCCGGACACTGTGAAGGGCCGCTTCACCATCTCCCGTGACAATTCCCGCAACACTCTGGATCTTCAAATGAACAGTCTGCGCGTCGAGGACACGGCTGTCTATTATTGTGCGCGTCATAGTGGCTACGGTAGTAGCTACGGGGTTTTGTTTGCTTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCCAGATGACTCAGTCTCCGTCCTCCCTGTCTGCATCTGTAGGAGACCGCGTCACCATCACCTGCCGTGCAAGTCAGGACATTAGCAATTATTTAAACTGGCTTCAACAGAAACCGGGGAAAGCCCCGAAGCTCCTGATTTACTACACATCAATCTTACACTCAGGAGTCCCGTCACGCTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCGGAAGATTTTGCAACTTATTACTGTCAACAGGGTAATACGCTTCCGTGGACGTTTGGCCAGGGGACCAAACTGGAAATCAAACGTTCGGATCCAGCCGAACCAAAGAGCCCCGATAAGACCCACACCTGTCCCCCCTGCCCAGCCCCAGAGCTGCTGGGAGGCCCCAGCGTGTTTCTGTTTCCACCCAAGCCAAAGGATACCCTGATGATTAGTAGAACACCCGAAGTGACCTGTGTGGTGGTGGATGTGTCTCACGAGGACCCCGAGGTGAAATTTAATTGGTATGTTGATGGTGTTGAAGTGCACAACGCCAAAACCAAACCCAGAGAGGAGCAGTACAATTCTACCTATAGAGTCGTGTCTGTGCTGACAGTGCTGCATCAGGATTGGCTGAACGGAAAAGAATACAAATGTAAAGTGAGCAATAAGGCCCTGCCCGCTCCAATTGAGAAGACAATTAGCAAGGCCAAGGGCCAGCCAAGGGAGCCCCAGGTGTATACACTGCCACCCAGTAGAGACGAACTGACAAAGAATCAGGTGTCTCTGACATGTCTGGTGAAGGGATTTTACCCATCTGATATCGCCGTGGAATGGGAATCTAACGGCCAGCCCGAGAATAACTATAAGACAACCCCACCAGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATTCTAAGCTGACAGTGGATAAGTCCCGGTGGCAGCAGGGAAATGTGTTTAGCTGTAGTGTCATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGTTCTGGGTCCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCTCTCCTGGTGACCGTGGCCTTCATCATCTTTTGGGTGCGCTCCCGGGTGAAGTTTTCTCGCTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAATCAGCTGTACAATGAACTGAACCTGGGCAGGCGGGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCACGGCGCAAAAATCCCCAGGAGGGACTCTATAACGAGCTGCAGAAGGACAAAATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGAAGACGCGGAAAGGGCCACGACGGCCTGTATCAGGGATTGTCCACCGCTACAAAAGATACATATGATGCCCTGCACATGCAGGCCCTGCCACCCAGAT GA SEQ ID NO: 14MSLPVTALLLPLALLLHAARPYPYDVPDYASLSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSNWMHWVRQAPGQGLEWMGEIDPSDSYTNYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARGSNPYYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSGVNYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSLEPEDFAVYYCHQRGSYTEGGGTKLEIKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQVPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNSRNTLDLQMNSLRVEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWLQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

To demonstrate co-expression of both CARs, the scFv of each CAR wastagged with an epitope tag (HA or V5 respectively). This subsequentsingle open-reading frame was cloned into the SFG retroviral vector.T-cells were transduced with this vector and both CARs could be detectedon the T-cells surface expressing the cassette by staining with anti-HAand anti-V5 and studying expression by flow cytometry.

Next, T-cells expressing the CD19 OR CD22 CAR gate were challenged withtarget cells, expressing neither, both or one antigen along with controlT-cells which expressed no CARs, or just anti-CD19 CAR alone, oranti-CD22 CAR alone. We found that the OR-gated CAR T-cells could killtarget cells expressing either one or both target antigens (FIG. 5).

Example 2 Identification and Characterisation of CD19ALAb and CD22ALAb

A CD19-binder (CD19ALAb) was identified, humanised and the bindingaffinities of both murine and humanised IgGs and scFvs were identifiedand compared with the “gold-standard” anti-CD19 binder, fmc63. Inparallel, and a CD22-binder (CD22ALAb) was identified, humanised and thebinding affinities of both murine and humanised IgGs and scFvs wereidentified and compared with the “gold-standard” anti-CD22 binder, M971.

Experiments were performed on a Biacore T200 instrument using HBS-P asrunning and dilution buffer. BlAevaluation software Version 2.0 was usedfor data processing. For binding kinetics, mouse anti-human IgG or goatanti-mouse IgG was covalently coupled to a CM5 Sensor Chip. IgG orscFv-Fc proteins were captured, and various concentrations ofinteraction partner protein injected over the flow cell at a flow rateof 30 μl/min. Kinetic rate constants were obtained by curve fittingaccording to a 1:1 Langmuir binding model. Bulk refractive indexdifferences were subtracted using a blank control flow cell in whichcapture antibody had been immobilized to the same level as the activesurface. A double reference subtraction was performed using bufferalone.

The results are shown in FIGS. 6 to 8.

The data show that humanised CD22ALAb has comparable binding affinity toCD22 to murine CD22ALAb (FIG. 6) and similar binding kinetics. Bothmurine and humanised CD22ALAb in an scFv format have significantlyhigher binding affinity to CD22 than the gold-standard CD22-bindingantibody, M971 (FIG. 6).

Although the binding affinity of murine and humanised CD19ALAb in an IgGformat was found to be similar (data not shown), surprisingly thebinding affinity of humanised CD19ALAb was found to be higher thanmurine CD19ALAb in an scFv format (FIG. 7). The binding affinity ofCD19ALAb is comparable (possibly slightly better) than that of thegold-standard anti-CD19 Ab, fmc63 (FIG. 8).

Example 3 Comparative Functional Assays With CD19ALAb/fmc63 CARs andCD22ALAb/M971 CARs

The antigen binding domain of a CAR can affect its function. In thisstudy, CARs were created comprising CD19ALAb and CD22ALAb and functionwas compared with an equivalent CAR having an antigen-binding domainbased on fmc63 or M971.

CARs comprising scFvs based on fmc63 (anti-CD19) and M971 (anti-CD22)can be considered as the gold standard antibodies as both CARs are inclinical development.

CARs were constructed and expressed based on CD19ALAb, fmc63, CD22ALAband M971. Their structure is shown in FIG. 9. The CARs differed solelyin their antigen binding domain. In all constructs, the binding domainswere linked to the membrane with a CD8 stalk spacer and containedintracellular activatory motifs from 41BB and CD3-zeta.

Retroviruses were produced by transient transfection of 293T cells withplasmids encoding the CARs, gag/pol and the envelope protein RD114.After 3 days the supernatants were harvested and used to transducePHA/IL2-activated PBMCs with equal titres of retrovirus onretronectin-coated plates. Six days post-transduction CAR-expression wasconfirmed by flow cytometry and PBMCs were co-cultured in a 1:1 ratiowith either CD19+ BFP SupT1 cells (fmc63 and CD19ALAb CARs) or CD22+ BFPSupT1 cells (M971 and CD22ALAb CARs). Target cell killing was assayedafter one and three days. Also after one and three days, supernatantswere removed and interferon-y levels were assayed by ELISA.

The results are shown in FIGS. 10 and 11.

As shown in FIG. 10, the CAR with a CD19ALAb antigen binding domain gavemore killing of CD19+ve target cells (FIG. 10) at both Dayl and Day 3,than the equivalent CAR with a fmc63 binding domain.

With regard to CD22, the CAR with a CD22ALAb antigen binding domain gavemore killing of CD22+ve target cells (FIG. 11a ) after three days thanthe equivalent CAR with an M971 binding domain. IFNy release wassignificantly higher with the CD22ALAb CAR than the M971 CAR after thesame time frame.

CARs having an antigen-binding domain based on CD19ALAb and CD22ALAbtherefore have improved properties in terms of target cell killing thanequivalent CARs based on fmc63 and M971.

The CD22ALAb result is particularly surprising, given the findingsreported in Haso et al (2013) as above. In that study, differentanti-CD22 CARs were made and tested, with binding domains based on theanti-CD22 antibodies HA22, BL22 and m971. HA22 and BL22 scFvs bind to Igdomain 3 of CD22, whereas m971 binds within Ig domain 5-7 of CD22 (seeHaso et al (2013) FIG. 2B). It was reported that the m971-derived CARshowed superior target cell killing activity than HA22-derived CAR,which finding is attributed to the importance of the CD22 epitopetargeted by the CAR (Haso et al (2013) page 1168, last full paragraph).It is concluded that targeting a membrane proximal domain of CD22 is“the key element” in developing a highly active anti-CD22 CAR(Discussion, last paragraph). Contrary to this finding, the data shownhere in FIG. 11 demonstrate that CD22ALAb, which targets an epitope inIg domain 3 of CD22—a “membrane distal” epitope compared to the Igdomain 5-7 epitope targeted by m971—has superior target cell killingability than an m971-based anti-CD22 CAR.

Example 4 Investigating OR Gate Constructs With Different EndodomainCombinations

Four OR gate constructs were developed as shown in FIG. 13. They allencoded CD19/CD22 OR gates having identical antigen-binding domains,spacer domains and transmembrane domains: the only difference betweenthe construct was in the endodomains, which were as shown in thefollowing Table:

CD19 CAR CD22 CAR Construct endodomain endodomain A 41BB-CD3ζ 41BB-CD3ζB OX40-CD3ζ OX40-CD3ζ C 41BB-CD3ζ CD28-CD3ζ D OX40-CD3ζ CD28-CD3ζ

The capacity of cells expressing each CD19/CD22 OR gate to kill Rajicells in vitro was assayed as described above. Transduced PBMCsexpressing the various OR gate combinations were co-cultured for 72hours with CD19+/CD22+ Raji target cells at both a 1:1 and 1:10effector:target cell ratio.

The results are shown in FIG. 14. All four OR gates were found to killtarget cells significantly better than the fmc63 and M971 CARs. With the1:10 effector:target cell ratio, it was shown that the “split”endodomain OR gates, which have 4-1BBzeta/OX40zeta on one CAR andCD28zeta on the other CAR, had the best killing activity.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, cell biology or related fields are intended to bewithin the scope of the following claims.

1-3. (canceled)
 4. A nucleic acid comprising a nucleotide sequenceencoding a first chimeric antigen receptor (CAR) and a nucleotidesequence encoding a second CAR as separate molecules, each CARcomprising: an antigen-binding domain; a spacer; a trans-membranedomain; and an endodomain, wherein the antigen-binding domain of thefirst CAR binds to CD19 and the antigen-binding domain of the second CARbinds to CD22.
 5. A nucleic acid sequence according to claim 4, whichhas the following structure:AuB1-spacer1-TM1-endo1-coexpr-AaB2-spacer2-TM2-endo2 in which AgB1 is anucleic acid sequence encoding the antigen-binding domain of the firstCAR; spacer1 is a nucleic acid sequence encoding the spacer of the firstCAR; CAR; TM1 is a nucleic acid sequence encoding the transmembranedomain of the first CAR; endol is a nucleic acid sequence encoding theendodomain of the first CAR; coexpr is a nucleic acid sequence enablingco-expression of both CARs; AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; cpaccr 2 spacer2 is a nucleicacid sequence encoding the spacer of the second CAR; TM2 is a nucleicacid sequence encoding the transmembrane domain of the second CAR; andendo2 is a nucleic acid sequence encoding the endodomain of the secondCAR; which nucleic acid sequence, when expressed in a T cell, encodes apolypeptide which is cleaved at a cleavage site such that the first andsecond CARs are co-expressed as separate molecules at the T cellsurface.
 6. A nucleic acid according to claim 5, wherein coexpr encodesa sequence comprising a self-cleaving peptide.
 7. A nucleic acidaccording to claim 5, wherein alternative codons are used in regions ofsequence encoding the same or similar amino acid sequences, in order toavoid homologous recombination.
 8. A kit which comprises a first nucleicacid that comprises a nucleotide sequence encoding a first chimericantigen receptor (CAR) and second nucleic acid that comprises anucleotide sequence encoding a second CAR, each CAR comprising: anantigen-binding domain; a spacer; a trans-membrane domain; and anendodomain, wherein the antigen-binding domain of the first CAR binds toCD19 and the antigen-binding domain of the second CAR binds to CD22,wherein the nucleic acid sequence of the first nucleic acid has thefollowing structure: AgB1-spacer1-TM1-endo1 in which AgB1 is a nucleicacid sequence encoding the antigen-binding domain of the first CAR;spacer1 is a nucleic acid sequence encoding the spacer of the first CAR;TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR; and endol is a nucleic acid sequence encoding the endodomainof the first CAR; and wherein the nucleic acid sequence of the secondnucleic acid has the following structure: AdB2-spacer2-TM2-endo2 inwhich AgB2 is a nucleic acid sequence encoding the antigen-bindingdomain of the second CAR; spacer2 is a nucleic acid sequence encodingthe spacer of the second CAR; TM2 is a nucleic acid sequence encodingthe transmembrane domain of the second CAR; and endo2 is a nucleic acidsequence encoding the endodomain of the second CAR.
 9. A kit comprising:a first vector which comprises a first nucleic acid comprising anucleotide sequence encoding a first chimeric antigen receptor (CAR) anda second vector which comprises a nucleic acid comprising a nucleotidesequence encoding a second CAR, each CAR comprising: an antigen-bindingdomain; a spacer; a trans-membrane domain; and an endodomain, whereinthe antigen-binding domain of the first CAR binds to CD19 and theantigen-binding domain of the second CAR binds to CD22.
 10. A kitaccording to claim 9, wherein the vectors are integrating viral vectorsor transposons.
 11. A vector comprising a nucleic acid according toclaim
 4. 12. A vector according to claim 11 which is a retroviral vectoror a lentiviral vector or a transposon.
 13. A method for making a cellwhich co-expresses a first chimeric antigen receptor (CAR) and secondCAR as separate molecules at the cell surface, each CAR comprising: anantigen-binding domain; a spacer; a trans-membrane domain; and anendodomain, wherein the antigen-binding domain of the first CAR binds toCD19 and the antigen-binding domain of the second CAR binds to CD22, themethod comprising a step of introducing: a nucleic acid according toclaim 4 into a cell, or introducting a vector comprising said nucleicacid into the cell.
 14. A method according to claim 13, wherein the cellis from a sample isolated from a subject. 15-45. (canceled)
 46. Anucleic acid according to claim 6, wherein alternative codons are usedin regions of sequence encoding the same or similar amino acidsequences, in order to avoid homologous recombination.
 47. The nucleicacid according to claim 4, wherein the first CAR and the second CAR eachcomprises a CD3 zeta endodomain.
 48. The nucleic acid according to claim5, wherein the first CAR and the second CAR each comprises a CD3 zetaendodomain.
 49. The nucleic acid according to claim 4, wherein the firstCAR and the second CAR each comprises a compound endodomain comprised of(a) a CD3 zeta domain and (b) a co-stimulatory domain or a TNF receptorfamily endodomain.
 50. The nucleic acid according to claim 49, whereineach compound endodomain comprises a 41 BB endodomain, an OX40endodomain, or a CD28 endodomain.
 51. The nucleic acid according toclaim 5, wherein the first CAR and the second CAR each comprises acompound endodomain comprised of (a) a CD3 zeta domain and (b) aco-stimulatory domain or a TNF receptor family endodomain.
 52. Thenucleic acid according to claim 51, wherein each compound endodomaincomprises a 41 BB endodomain, an OX40 endodomain, or a CD28 endodomain.53. A method for making a cell which co-expresses a first chimericantigen receptor (CAR) and second CAR as separate molecules at the cellsurface, each CAR comprising: an antigen-binding domain; a spacer; atrans-membrane domain; and an endodomain, wherein the antigen-bindingdomain of the first CAR binds to CD19 and the antigen-binding domain ofthe second CAR binds to CD22, the method comprising a step ofintroducing a nucleic acid according to claim 5 into a cell, orintroducing a vector comprising said nucleic acid into the cell.
 54. Amethod for making a cell which co-expresses a first chimeric antigenreceptor (CAR) and second CAR as separate molecules at the cell surface,the method comprising introducing first and second nucleic acids intothe cell: wherein the first nucleic acid comprises a nucleotide sequencewith the following structure: AgB1-spacerl-TM1-endo1 in which AgB1 is anucleic acid sequence encoding the antigen-binding domain of the firstCAR; spacer1 is a nucleic acid sequence encoding the spacer of the firstCAR; TM1 is a nucleic acid sequence encoding the transmembrane domain ofthe first CAR; and endol is a nucleic acid sequence encoding theendodomain of the first CAR; and wherein the second nucleic acidsequence comprises a nucleotide sequence with the following structure:Ag B2-spacer2-TM2-endo2 in which AgB2 is a nucleic acid sequenceencoding the antigen-binding domain of the second CAR; spacer2 is anucleic acid sequence encoding the spacer of the second CAR; TM2 is anucleic acid sequence encoding the transmembrane domain of the secondCAR; and endo2 is a nucleic acid sequence encoding the endodomain of thesecond CAR, wherein the antigen-binding domain of the first CAR binds toCD19 and the antigen-binding domain of the second CAR binds to CD22. 55.A method for making a cell which co-expresses a first chimeric antigenreceptor (CAR) and second CAR as separate molecules at the cell surface,the method comprising introducing first and second vectors into thecell: wherein the first vector comprises a nucleic acid comprising anucleotide sequence encoding the first CAR and the second vectorcomprises a nucleic acid comprising a nucleotide sequence encoding thesecond CAR, each CAR comprising: an antigen-binding domain; a spacer; atrans-membrane domain; and an endodomain, wherein the antigen-bindingdomain of the first CAR binds to CD19 and the antigen-binding domain ofthe second CAR binds to CD22.
 56. A method according to claim 55,wherein the vectors are integrating viral vectors or transposons.